Having identified the p38a subproteome, we will next undertake analysis of phosphoproteins within this[unreadable] subproteome using a combination of 1DE/2DE, IMAC and selective ion scanning mass spectrometry methods.[unreadable] Specifically, p38a-associated proteins will be isolated by Flag pull-down, separated by electrophoresis, and digested[unreadable] with trypsin. These tryptic peptides will either be subjected directly to precursor ion/neutral loss scan analysis with[unreadable] mass spectrometry, or will first undergo a phosphopeptide enrichment step using IMAC. Putative phosphoprotein[unreadable] identifications will be verified by western immunoblotting. Detailed procedures for IMAC and precursor/neutral loss[unreadable] ion scanning are listed below.[unreadable] II.D. Collaboration with Project 4[unreadable] The general approach to characterize the Cdk2 subproteome will proceed via three steps (parallel to the[unreadable] analysis of p38a described above). First we will identify members of the subproteome by immunoprecipitation,[unreadable] electrophoresis and LC/MS/MS. Second, we will determine quantitative changes in this subproteome in the ischemic[unreadable] heart using DIGE and ICAT technologies. Third, we will examine phosphorylated members of this subproteome[unreadable] using IMAC and selective ion scanning methods.[unreadable] Aim 1 of Project 4 will examine proteins associated with Cdk2 in the normal and ischemic myocardium using[unreadable] a combination of immunoprecipitation (IP), 1DE/2DE, and LC/MS/MS. The mitochondrial fraction will be[unreadable] resuspended in sucrose buffer, sonicated briefly, and treated with 0.1% DDM (see Sample Preparation below for[unreadable] specific methodology). The mixture will be centrifuged again to pellet mitochondrial membranes, and released[unreadable] mitochondrial proteins in the supernatant will be used for IP. Myocardial cytosolic or mitochondrial fractions will be[unreadable] pre-cleared with protein A/G beads and Cdk2-associated proteins will be isolated by IP with anti-Cdk2 monoclonal[unreadable] antibodies (Santa Cruz, sc-6248). IgG will be used in place of anti-Cdk2 antibody as a negative control. Cdk2-[unreadable] associated proteins, retained on the beads after washing to remove non-specific interactions, will be eluted from the[unreadable] beads and separated by 1DE. Proteins will be excised from the gel, trypsinized and subjected to LC/MS/MS[unreadable] analysis. [Note: The members of Project 4 have worked closely with the Proteomic Core to optimize the purification[unreadable] of Cdk2 complexes by IP and the running of 1DE/2DE gels and will continue to do so for the duration of these[unreadable] studies.] Together, these techniques will provide the first map of the Cdk2 subproteome in the heart. Detailed[unreadable] procedures for 1DE and LC/MS/MS are described below. See Figure 2 for a general overview of the experimental[unreadable] strategy for Projects 3 and 4.[unreadable] Like p38a, Cdk2 is a soluble kinase, and thus we may be able to gain significant information from 2DE[unreadable] separation of Cdk2 complexes, especially those isolated from the cytosol. Also analogous to p38a, we will[unreadable] implement DIGE and ICAT technologies for the quantitation of protein abundance changes within the Cdk2[unreadable] subproteome. We will also utilize IMAC and selective ion scanning to determine phosphorytation events within the[unreadable] Cdk2 subproteome and to track changes in these modifications in the ischemic myocardium. Extensive methodology[unreadable] for these techniques is provided below.[unreadable] PHS 398 (Rev. 05/01) Page 321[unreadable] Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.[unreadable] CONTINUATION PAGE Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Vondriska, ProteomJC Core)[unreadable] Note: Although the initial analyses of the p38a and Cdk2 subproteomes will employ denaturing techniques[unreadable] on the front end (1DE and 2DE), we are committed to comprehensive characterization of these subproteomes using[unreadable] nondenaturing approaches (such as BN PAGE) as described in Project 1 with VDAC. After the initial[unreadable] characterization of the p38a and Cdk2 subproteomes in years 1and 2 of the PPG, we will analyze native complexes[unreadable] formed by these molecules with a more directed mass spectrometry and immunoblotting approaches in years 3-5 of[unreadable] the PPG.[unreadable] III. VALIDATION STEPS[unreadable] III.A. Quantitative Validation of Immunoprecipitations and Affinity Pull-Downs and Necessary Controls[unreadable] We will perform western immunoblotting experiments to ensure that the pull-downs to isolate the[unreadable] subproteomes of all molecules are quantitatively consistent. Specifically, we will immunoblot for the target of[unreadable] isolation to ensure that the same amount of protein is used under all conditions to isolate protein complexes. For[unreadable] example, in Project 3, we will western blot for p38a on equal-volume amounts of all the Flag pull-downs from[unreadable] different isolations to be certain that changes in protein association with p38a cannot be attributed to changes in the[unreadable] amount of p38a isolated (i.e. the amount of Flag-p38a pulled down in each experiment will be constant). Likewise in[unreadable] Project 4, we will immunoblot for Cdk2 following immunoprecipitation of Cdk2 based on the same principle. In this[unreadable] regard, we can be sure that the amount of Cdk2 isolated in different experiments, and between different treatment[unreadable] groups, is equal, and thus changes in the associating proteins cannot be attributed to changes in the amount of[unreadable] target protein isolated.[unreadable] A second issue of considerable importance is the use of essential control groups for isolation of protein[unreadable] complexes by immunoprecipitation or affinity pull-down. In both cases, proteins samples are incubated with beads[unreadable] alone, prior to addition of antibody or recombinant protein, to "preclear" the lysate of non-specific interactions[unreadable] between sample proteins and the beads. In the case of immunoprecipitation, a second control that is always[unreadable] performed is substitution of the IgG for the antibody against the target of isolation (e.g. when Cdk2-associated[unreadable] proteins are isolated in Project 4, we will perform parallel experiments using mouse IgG in place of the Cdk2[unreadable] antibody). This step is required to exclude proteins that associate with the nonspecific region of the antibody. For[unreadable] affinity pull-down experiments (e.g. isolation of VDAC native complexes in Project 1), the second control in addition[unreadable] to preclearing with beads is the substitution of HIS-null proteins for HIS-VDAC. This step helps exclude from[unreadable] analysis proteins that associate with the HIS epitope tag.[unreadable] III.B. Functional Validation of Subproteome Candidates: Collaboration with Heart Biology Core[unreadable] In addition to interacting with the individual Projects to perform proteomics experiments, the Proteomic Core[unreadable] will also interact extensively with the Heart Biology Core to facilitate verification of candidate members of the[unreadable] subproteomes. The validation process involves a battery of classical biochemistry, cell and organelle biology and[unreadable] histo-pathology approaches to determine the functional role of the individual molecules in the context of the entire[unreadable] subproteome in vivo. It is important to highlight the close interaction between the Heart Biology Core and the[unreadable] Proteomic Core that will persist throughout this PPG.[unreadable] In Project 1, we will identify the PKCe-VDAC and PP2CK-VDAC associated proteins and generate a[unreadable] bioinformatic map of these subproteomes in the context of the MPT pore as a whole. Once we have identified the[unreadable] members of these subproteomes, we will confirm these findings by testing for co-localization of the candidate[unreadable] molecules with VDAC and other core components of the MPT pore (such as ANT) in the heart using confocal[unreadable] microscopy in collaboration with the Heart Biology Core. When available, we will also use inhibitors and/or activators[unreadable] of these molecules to examine the effects on mitochondrial function. In Project 2, we will determine residues on[unreadable] ANT1 and VDAC that are targeted for phosphorylation in the protected myocardium. Once these residues have[unreadable] been identified, we will collaborate with the Heart Biology Core and Project 2 to test the functional role of these[unreadable] residues using reconstitution assays and genetic manipulation. In Projects 3 and 4, we will identify p38a- and Cdk2-[unreadable] associated proteins, respectively, and will incorporate this subproteomic data with the PKCe-VDAC/PP2Cic-VDAC[unreadable] bioinformatic map being generated in Project 1. Because we will also map cytosolic and mitochondrial complexes in[unreadable] Projects 3 and 4, the findings will further flesh out any role for p38a- or Cdk2-associated proteins in the regulation of[unreadable] the mitochondria from an "inside-out" and "outside-in" perspective. As with Project 1, the Proteomic Core will[unreadable] collaborate heavily with the Heart Biology Core in Projects 3 and 4 to confirm the localization of p38a- or Cdk2-[unreadable] associated proteins to the mitochondria, and to examine colocalization of these molecules with the components of[unreadable] PHS 398 (Rev. 05/01) Page 322[unreadable] Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b.[unreadable] CONTINUATION PAGE Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Vondciska, PrOteOmJC Core)[unreadable] the MPT pore (such as VDAC and ANT) using confocal microscopy. When available, we will also use inhibitors[unreadable] and/or activators of these molecules to examine the effects on mitochondrial function.[unreadable] Overall, there is a bidirectional flow of information and collaboration between the two Cores in this Program[unreadable] Project. As the Proteomic Core unveils novel members of a subproteome, the Heart Biology Core will provide the[unreadable] resources to the individual Projects to test the functional role of these candidates in the cardiac cell. Likewise, as the[unreadable] Heart Biology Core and the individual Projects decipher new information about the functional aspects of these[unreadable] subproteomes, the Proteomic Core will investigate novel protein complexes to further expand our understanding of[unreadable] the signal network. In this regard, the interaction between the Proteomic Core and the Heart Biology Core will[unreadable] aid execution of the experiments proposed in Projects 1-4, however, this interaction will also generate novel[unreadable] hypotheses about the cardioprotective signaling network to be tested in the future.[unreadable]